Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 A resolution. The structure reveals that the translocation domain contains a central pair of alpha-helices 105 A long and a approximately 50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site--a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.

SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinurn neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln'97-Arg'98 peptide bond, while serotype E cleaves the Arg'80-Ile'8' peptide linkage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltagedependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.Diphtheria toxin (1), botulinum neurotoxin (2), and tetanus toxin (3) are proteins that are similar in origin and macrostructure. All three toxins are synthesized by bacteria (Corynebacterium diphtheriae, Clostridium botulinum, and Clostridium tetani) as single polypeptide chains (diphtheria toxin, "'60 kDa; clostridial neurotoxins, ==150 kDa). When exposed to trypsin or trypsin-like enzymes, they are cleaved to yield two-chain molecules in which a heavy-chain polypeptide is linked by a disulfide bond to a light-chain polypeptide. The two-chain structure is the active form of the three toxins.Various techniques have been used to generate polypeptide fragments from the toxins. The most straightforward of these is disulfide-bond reduction, which releases the heavy chain from the light chain. Alternatively, the clostridial neurotoxins have been exposed to limited proteolysis (e.g., with papain) to generate a fragment B and fragment C. Finally, traditional techniques have been used to select mutant organisms that synthesize incomplete toxins, such as the CRM45 fragment of diphtheria toxin, from which the B45 fragment can be formed. The various toxins and their fragments are illustrated in Fig. 1 Previous studies have shown that the amino terminus of the heavy chain from diphtheria toxin (18) and whole diphtheria toxin (19) form channels in lipid bilayers, and it has been proposed (18) that these channels provide the pathway for the light chain to cross membranes. Here we report that the heavy chains of both botulinum neurotoxin type B and tetanus toxin also form channels in lipid bilayers. Furthermore, for all three toxins, channel formation is maximal when the protein-containing (cis) side of the artificial membrane is at low pH (-4.0) and the opposite (trans) side is at pH -7.0, a pH gradient comparable to that across the membranes of acidic vesicles in cells. The channels for all three toxins are very large, as determined by selectivity experiments with large anions and cations, and this finding is compatible with the idea that the channels function as "tunnel proteins" for translocation of fully extended active fragments. In addition, tetanus toxin channels display a voltage dependence similar to that of diphtheria toxin channels, opening when positive voltages are applied to the cis side of artificial membranes and closing when nega...

An ATP-dependent activity of NSF (N-ethylmaleimidesensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca 2؉ -triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with ␣-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca 2؉ -triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSFcatalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca 2؉ -dependent mechanisms.The regulated fusion of vesicles with the plasma membrane in neural and endocrine cells requires a core complex of proteins (synaptobrevin, syntaxin, and SNAP-25) that are specific substrates for clostridial neurotoxin proteases (1-4). This complex is proposed to function in vesicle targeting, docking or fusion. Identification of these neuronal synaptic proteins (termed SNAREs) 1 as receptors for SNAP proteins that mediate the membrane association of NSF, a protein required for constitutive membrane fusion (1), suggested that NSF may be required for Ca 2ϩ -regulated neurosecretion (5). Genetic studies in Drosophila have established an essential role for NSF in neural function (6). Stimulatory effects of ␣-SNAP on neurotransmitter secretion from chromaffin cells and squid neurons have been reported (7,8). However, the precise stage in the regulated secretory pathway at which NSF acts has not been directly established. In vitro biochemical studies demonstrated that a 20 S complex of SNAREs, NSF, and ␣/␤-SNAP was disassembled by the ATP-dependent activity of NSF, and it was suggested that NSF-catalyzed SNARE protein rearrangements drive membrane fusion (9). However, previous studies with permeable PC12 and adrenal cells had shown that MgATP was required for a priming step that precedes the final fusion steps triggered by Ca 2ϩ (10,11). In the present studies, the execution point of NSF and ␣-SNAP was established as the ATP-dependent priming step that precedes Ca 2ϩ -activated fusion. EXPERIMENTAL PROCEDURES Preparation of Permeable PC12 Cells and Secretion Assays-PC12 cells were labeled with [3 H]norepinephrine (NE; Amersham Corp.) and permeabilized with a ball homogenizer (10, 12). Two stage secretion assays were in KGlu buffer (20 mM HEPES, pH 7.2, 120 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) with 0.1% bovine serum albumin. Thirty-min priming incubations at 30°C contained 2 mM MgATP and 1.0 mg/ml rat brain cytosol, whic...